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Review Therapy for fungal diseases: opportunities and priorities

David W. Denning and William W. Hope

School of Translational Medicine, Manchester Academic Health Science Centre, University of Manchester, University Hospital of South Manchester, Manchester M23 9LT, UK

This article provides a perspective on the current status Currently, five classes of agents are used of drug therapy for invasive fungal diseases, together orally or intravenously for the treatment of fungal with priorities for the future development of novel com- in humans: polyenes, pyrimidine analogues, , pounds. Key opportunities for new drugs include pro- and the (Table 1). Each antifungal duction of orally bioavailable agents for the treatment of compound has advantages and limitations related to its invasive , invasive , cryptococcal spectrum of activity (Table 2), route of administration, drug meningitis and mucosal and urinary infections. interactions and toxicity profile. This review will describe Orally bioavailable agents for the treatment of chronic the role and limitations of these agents for the treatment of pulmonary and allergic aspergillosis are also required, as the most common and medically important syndromes. well as new potent drugs against a range of medically Agents that are in early phases of development will not important moulds. Antifungal resistance is a problem in be discussed. We reflect on the challenges and opportunities certain contexts, but is generally less of a problem than for the development of novel therapeutic strategies for the bacterial infections. Earlier and more complete myco- most common fungal syndromes as a way of improving logical diagnosis and improvements in underlying risk outcomes. Our key insights for drug development and future estimation will improve outcomes. The limitations of the research are summarised in Box 1 and Box 2. current antifungal agents and opportunities for new developments are discussed. Antifungal compounds Polyenes Fungal infections: the challenges ahead The polyenes are broad-spectrum antifungal agents pro- Invasive fungal infections are an increasing threat to duced by the bacterial genus Streptomyces. was human health. In the developed world, these infections discovered in 1950 from the fermentation broth of Strep- predominantly occur in the context of increasingly tomyces noursei, and is still used as a topical antifungal aggressive immunosuppressive therapies. The overall agent. This was followed by isolation of fracidin, rimocidin, mortality for invasive diseases caused by Candida spp. endomycin, ascosin, trichomycin and antimycoin in the and spp. is 30–50%, despite the advent of early 1950 s [5]. Amphotericin A and B were isolated from new diagnostic and therapeutic strategies. In the devel- S. nodosum and reported in 1955 [6], but only amphoter- oping world, there are 1 million cases of cryptococcal icin B was developed because of its superior potency. Since disease per year, resulting in 675 000 deaths [1]. Allergic then, 90 polyenes have been discovered, but problems fungal syndromes are increasingly recognised [2].Con-  with solubility, stability, oral bioavailability and toxicity tinued efforts are required to improve the often subopti- have prevented many of these compounds being developed mal therapeutic outcomes associated with fungal for clinical purposes [5]. is still widely used as a infections. topical ophthalmic agent. The high degree of phylogenetic relatedness between The complex chemistry and systemic toxicity of the poly- fungi and humans means that there are relatively few enes provided the impetus to develop novel drug delivery differential targets to be exploited for antifungal drug systems that enable systemic therapy [7]. The best example development. Fungi are involved in an interminable of this is the lipid formulations of , although a struggle for survival with each other and with other liposomal formulation of nystatin was also developed [8]. microbes. They produce a vast array of extracellular The current commercially available lipid preparations of enzymes and secondary metabolites to counteract and amphotericin B include amphotericin B lipid complex digest the external world. Many antimicrobial agents have (ABLC), amphotericin B colloidal dispersion (ABCD) and been isolated from fungi themselves [3]. The best example liposomal amphotericin B (Figure 1). Other formulations are is penicillin, which was isolated from Penicillium notatum in various stages of clinical development, including prep- (now Penicillium chrysogenum) by Fleming, and later pur- arations that are orally bioavailable [9]. Lipid formulations ified for medical use by Florey and Chain [4]. Similarly, the differ significantly in terms of their pharmacokinetics, tissue echinocandins, a novel class of antifungal compounds now distribution and toxicity profile [10,11].AmphotericinB in widespread clinical use, are semisynthetic derivatives of formulations are widely used for the treatment of dissemi- fungal-derived cyclic hexapeptides. nated candidiasis, invasive aspergillosis, cryptococcal Corresponding author: Denning, D.W. ([email protected]). meningitis, and infections caused by the [12,13].

0966-842X/$ – see front matter ß 2010 Published by Elsevier Ltd. doi:10.1016/j.tim.2010.02.004 Available online xxxxxx 1 TIMI-711; No. of Pages 10

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Table 1. Characteristics of antifungal agents used to treat invasive or allergic diseasea Agent Routes of Frequency of Adverse events, Clinically relevant Comments administration administration toxicity drug–drug interactions Polyenes Amphotericin Ba IV, Nebulised Once daily Infusional toxicity, Renal impairment Lipid formulations associated with less nephrotoxicity, indirectly affects infusional toxicity and low blood potassium action of many nephrotoxicity drugs Pyrimidine analogues po, IV 1–4 times daily Bone marrow None Used for induction therapy for depending on suppression, cryptococcal meningitis; only renal function deranged LFT results in combination with another and blood levels antifungal agent; TDM necessary Azoles IV, po Once daily Gastrointestinal Few Some resistance, narrow intolerance, spectrum deranged LFT results po, IV Twice daily Fluid retention, Numerous, due to Oral bioavailability depends left ventricular inhibition of CYP 3A4b on specific formulation, dysfunction, with variability between gastrointestinal generic preparations; intolerance TDM ideally required IV, po, ocular Twice daily Deranged LFT Numerous, due to Nonlinear pharmacokinetics results, inhibition of CYP3A4c; and substantial inter-individual photosensitive some interactions with variability in serum concentrations; rash, altered CYP 2C19 metabolism very rapid metabolism in children; vision, hallucinations, (e.g. omeprazole) TDM ideally required confusion po 2–4 times daily Gastrointestinal Some, due to Other formulations in development; intolerance, inhibition TDM ideally required deranged LFT results of CYP3A4c Allylamines Terbinafine po Once daily Deranged LFT results, Few Used for fungal nail infections. mild rash, nausea, Not of use for invasive loss of taste Echinocandins IV Once daily Phlebitis, nausea, Ciclosporin, Modest efficacy as first-line agent deranged LFT results rifampicin for invasive aspergillosis (?in combination with other drugs) IV Once daily Phlebitis, nausea, Few Modest efficacy as first line agent deranged LFT results for invasive and chronic aspergillosis IV Once daily Histamine-like Few No data reported for aspergillosis reactions, diarrhoea, deranged LFT results Abbreviations: CYP 3A4, Cytochrome P450 isozyme 3A4; IV, intravenous; LFT, liver function test; po, oral; TDM, therapeutic drug monitoring. aAmphotericin B deoxycholate, liposomal amphotericin B, amphotericin B lipid complex or amphotericin B colloid dispersion. bThe compound inhibits CYP 3A4 and leads to higher concentrations of drugs metabolised by this enzyme; low concentrations with compounds that accelerate metabolism through CYP 3A4 (e.g. rifampicin, anticonvulsants).

Pyrimidine analogues interfere with DNA and RNA synthesis. [16]. The differ- Flucytosine (5-fluorocytosine; 5FC) was discovered in 1964 ential antifungal activity of flucytosine results from the by Roche Laboratories within an anti-neoplastic drug de- absence of cytosine deaminase in humans (Figure 2). velopment programme. Flucytosine does not have any In vitro studies described the emergence of resistance to intrinsic anti-neoplastic activity, but is active against flucytosine with concentrations < 25 mg LÀ1 [17], leading medically important such as Candida spp. and to the longstanding dogma that flucytosine should always Cryptococcus spp., and against a limited number of moulds be used in combination. Flucytosine causes bone marrow such as Aspergillus spp. and agents of chromoblastomyco- suppression, especially with peak plasma concentrations sis [14,15]. that are persistently >100 mg LÀ1, which is the primary Flucytosine is a , which acts as a reason why therapeutic drug monitoring is essential [18]. subversive substrate within the pyrimidine salvage path- Flucytosine (in combination with another antifungal way that is responsible for scavenging precursors for agent) is the standard of care for the treatment of crypto- nucleic acid synthesis. Flucytosine is transported into coccal meningitis [19], but is also occasionally used (in fungal cells by cytosine permeases, where it is then dea- combination) for the treatment of at minated to 5-fluorouracil (5FU) by a fungal enzyme, cyto- sites where drug penetration might be suboptimal (e.g. the sine deaminase. Sequential phosphorylation of 5FU eye or central nervous system), or to treat infections that produces toxic fluorinated nucleotides that ultimately are resistant or refractory to first-line antifungal agents.

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Table 2. Relative in vitro and clinical activity of antifungal agents for more common and important invasive fungal pathogensa Polyenes Echinocandins Amphotericin Fluconazole Itraconazole Voriconazole Posaconazole Caspofungin Micafungin Anidulafungin B formulations Candida spp. C. albicans ++ ++ ++ ++ ++ ++ ++ ++ C. glabrata ++ +/À N+ + ++++++ C. tropicalis ++ ++ ++ ++ ++ ++ ++ ++ C. parapsilosis ++ ++ ++ ++ ++ + + + C. krusei +-+/À + + ++ ++ ++ ++ + + ++ ++ ÀÀÀ spp. À ++ N N N ÀÀÀ Aspergillus spp. A. fumigatus ++ À ++ ++ ++ + + N A. flavus ++ À ++ ++ ++ + + N A. terreus ÀÀ++ ++ ++ + + N A. niger ++ À ++ ++ ++ + + N A. nidulans ÀÀ++ ++ ++ + + N Scedosporium prolificans ÀÀNN N ÀÀÀ Scedosporium apiospermum ÀÀ+/À +NÀÀÀ spp. + À -+ + ÀÀÀ Mucorales + À +/ÀÀ + ÀÀÀ Relative activity is indicated as follows: À, no activity; +/À, slight activity; +, modest activity; ++, good activity; N = no data. aMost fungi causing cutaneous and allergic disease have been excluded.

The and triazoles current clinical use include fluconazole, itraconazole, vor- , the first , was discovered in 1944 and iconazole and posaconazole; these are discussed below. demonstrated to be active against yeasts and several The putative antifungal target for the azoles is the bacterial species. Approximately 100 derivatives of this enzyme 14-a demethylase. The common or compound were subsequently synthesised. Several dec- ring (Figure 2) interacts with the haem moiety ades later, drug discovery programmes at Bayer led to of this enzyme; whereas, the side chain orientates the the discovery of the tritylimidazole derivative . active site of the molecule to the haem moiety within Phenethyl imidazole derivatives developed by Janssen the target protein. The interaction of the side chain within yielded , and . Ketoco- the binding pocket is responsible for the differential anti- nazole was the first orally bioavailable imidazole [20]. fungal activity that is characteristic of these compounds. Despite hepatotoxicity, inhibition of endogenous human Inhibition of 14-a demethylase disrupts the synthesis of cortisol synthesis and a multitude of drug interactions, sterols that are required for normal fungal membrane ketoconazole revolutionised the treatment of oral candi- function. diasis, , endemic mycoses, dermato- phyte infections and veterinary fungal infections. Fluconazole Fluconazole exhibits linear pharmacokinetics Subsequently, many azoles for agricultural, veterinary (i.e. the dose is directly proportional to the area under the and human use were developed. Modifications of the active concentration-time curve) [21], is water-soluble, and is ring produced the triazoles, and members of this class in very well tolerated. Despite its frequently reduced or absent activity against and the Box 1. Priorities for development of novel antifungal agents inherent resistance of Candida krusei, along with the for the treatment of invasive fungal infections absence of activity against Aspergillus spp. and other medically important moulds, fluconazole is extensively used for the prevention and treatment of superficial and  Oral compound with activity against all common Candida species invasive Candida infections [22,23]. Fluconazole is also (including triazole-resistant strains).  Parenteral or oral compounds with activity against Cryptococcus used for the treatment of cryptococcal infections and has neoformans and penetration into the central nervous system. demonstrated efficacy against [24].  Parenteral and oral compounds with activity against Aspergillus spp., including triazole-resistant species. Ideally, compounds Itraconazole Itraconazole was the first orally bioavailable should have few drug interactions, and should be safe in patients with renal or hepatic impairment. agent with anti-Aspergillus activity [25]. Human  Parenteral and oral compounds with activity against rare, but metabolism of itraconazole produces a metabolite, medically important moulds (e.g. Mucorales, Scedosporium spp.). hydroxyitraconazole, which has comparable antifungal  Oral agent(s) for the treatment of chronic pulmonary and allergic activity to the parent. Itraconazole is available as aspergillosis, with few drug interactions (especially with corticos- capsules for oral use, and as a cyclodextrin formulation teroids) and favourable intrapulmonary pharmacokinetics.  Development of novel formulations of existing compounds that for oral and intravenous use. The major limitation of the have a more favourable pharmacokinetic properties (e.g. en- capsule formulation is its highly variable acid-dependant hanced oral bioavailability) oral bioavailability [26]. This often leads to subtherapeutic  Formulations that enable novel uses of existing compounds (e.g. concentrations in critically ill patients with aerosolisation) hypochlorohydria (elevated gastric pH). Itraconazole

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Box 2. Priorities for future research fungal to improve the for recipients of bone marrow transplants who have graft- outcomes of invasive fungal infections versus-host disease [36,37]. This compound is also licensed for second-line therapy for patients with invasive asper-  Improved diagnostic tests, with high accuracy, rapid turnaround gillosis [38], and is partially effective for the treatment of time, and prognostic value that can guide antifungal therapy in infections caused by the Mucorales [39]. Posaconazole is real time. usually well tolerated.  Direct detection of species causing infection and antifungal resistance, without having to first culture the . Allylamines  Better risk prediction models, including genetic risk factors to target surveillance, prophylaxis and rapid diagnostics more Terbinafine, the only in clinical use, was dis- appropriately. covered in a medicinal chemistry programme as a deriva-  Mechanisms to ensure the attainment of maximal antifungal tive of the topical antifungal naftifine, which was the effect as quickly as possible (e.g. combination therapy, therapeu- original compound of the allylamine class. The target for tic drug monitoring). terbinafine, squalene epoxidase, was identified in 1984  Novel immunomodulatory treatments to maximise antifungal effect and minimise immune-mediated damage and fibrosis. [40]. Terbinafine is the agent of choice for many dermato-  Collaborative national and international programmes for anti- phyte infections including [41]. Although it fungal resistance surveillance. has limited activity for treatment of invasive fungal dis- eases, terbinafine can be combined with voriconazole for the treatment of infections caused by Scedosporium proli- solution results in improved oral bioavailability, but is often ficans [42]. associated with gastrointestinal intolerance. The intravenous formulation circumvents problems with Echinocandins variable oral bioavailability, but is not widely used. The The echinocandins are semi-synthetic cyclic lipohexapep- administration of itraconazole solution (not capsules) and tides with molecular weights of 1200 Da; which were serum trough concentrations >0.5 mg LÀ1 (measured by originally derived from fungi [43]. Cilofungin was the first HPLC) decreases the incidence of invasive fungal used in clinical trials, but its development infections in patients with profound and prolonged was abandoned because of toxicity. Caspofungin, micafun- neutropenia [27]. Itraconazole is used for the treatment of gin and anidulafungin are active against Candida spp. and chronic pulmonary aspergillosis, allergic syndromes related Aspergillus spp., but few other fungi. The echinocandins to Aspergillus spp., onychomycosis, , have a unique mechanism of action via the non-competitive and [28]. Itraconazole has inhibition of the cell wall protein 1,3-b-D-glucan synthase activity against ,andcanbeusedfor (Figure 2). Decreased 1,3-b-D-glucan concentrations in the the treatment of both vulvovaginal or cell wall result in osmotic fragility and lysis of Candida caused by fluconazole-resistant strains, providing there is spp. Although the echinocandins do not kill Aspergillus no laboratory evidence of cross-resistance. Itraconazole spp., they induce morphological changes within these fungi has no dependable activity against the Mucorales, and decrease their propensity to cause pulmonary injury although on occasions successful treatment has been [44]. The currently licensed echinocandins are not orally documented [29]. The use of itraconazole is associated bioavailable. with a number of toxicities, which increase with increasing drug exposure [30]. Therapy for Candida syndromes Voriconazole, a structural congener of fluconazole, is Candida spp. are human commensals that can cause active against Aspergillus spp., Fusarium spp. and Can- superficial and invasive infection. Despite improved un- dida spp. (including the fluconazole-resistant or less derstanding of the pathogenesis of invasive disease and the susceptible species of C. glabrata and C. krusei). Vorico- advent of new diagnostic and therapeutic strategies, the nazole has no activity against the Mucorales, but is a first- attributable mortality has essentially remained line agent for the treatment of invasive aspergillosis, and is unchanged for the past few decades at 40% [45,46]. superior to amphotericin B deoxycholate for initial therapy of this syndrome [31]. Voriconazole is not currently Disseminated candidiasis licensed for the prophylaxis of fungal infection, or for Disseminated candidiasis (candidaemia and invasive can- treatment of persistently febrile patients with low neutro- didiasis) is a life-threatening syndrome with an attribu- phil numbers who have suspected, but unproven infection table mortality of 10–50% [46]. Although amphotericin B [32]. Its clinical use is complicated by a number of drug deoxycholate is an effective agent for this syndrome, its use interactions, highly variable pharmacokinetics and more is limited by both infusional toxicity and nephrotoxicity. side effects than those of fluconazole. Liposomal amphotericin B is one of the first-line agents for Posaconazole is structurally related to itraconazole, and the treatment of disseminated candidiasis [13]. has broad spectrum activity against Candida spp., Asper- Fluconazole has been extensively used for the treatment gillus spp. and the Mucorales [33]. Oral bioavailability is of this syndrome, but since the introduction of the echino- increased by food, especially by fatty food [34,35]. Capsule candins, its role as a first-line agent is less clear. A recent and intravenous preparations are in development. There clinical trial comparing anidulafungin with fluconazole are fewer drug interactions compared with itraconazole or suggested that anidulafungin results in superior outcomes voriconazole. Posaconazole is effective for preventing fun- at the end of therapy [47]. First-line use of fluconazole is gal infections for neutropenic patients with leukaemia and reasonable for patients who (i) are haemodynamically

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Figure 1. The structures of selected antifungal agents within each of the major antifungal classes. stable; (ii) have not been previously exposed to triazoles; result of simple colonisation of the lower renal tract, of and (iii) have a low estimated risk of infection with C. upper renal tract disease or of ‘spill’ from bloodstream glabrata and C. krusei based on local epidemiological infection [58]. The majority of patients with asympto- patterns. Fluconazole can be used after initial parenteral matic candiduria do not require antifungal therapy. therapy in many patients to complete 2 weeks of antifungal Removal (or replacement) of an indwelling urinary therapy after negative blood cultures. Voriconazole is an catheter, if present, is usually effective [59].Iftherapy effective agent for disseminated candidiasis [48], but its is deemed necessary, fluconazole is the agent of choice use is often limited by drug interactions and (in the case of [60,61]. Bladder washouts with amphotericin B are an the intravenous formulation) accumulation of the cyclo- alternative for fluconazole-resistant infections. Flucyto- dextrin excipient in patients with a glomerular filtration sine achieves high urinary concentrations, but relapse is rate of < 50 mL minÀ1. Nevertheless, voriconazole might common. If candiduria is a manifestation of (suspected or have activity against fluconazole-resistant C. glabrata and proven) candidaemia, systemic therapy is warranted, C. krusei, and is a potential agent for oral therapy in this even if the chosen compound does not achieve high urin- circumstance. ary concentrations. Safe and well-tolerated agents with The echinocandins are increasingly used for the treat- activity against Candida in the urinary tract would be ment of disseminated candidiasis. They are as effective as useful for the modest number of patients for whom amphotericin B [13,49] and probably superior to flucona- specific therapy is indicated. zole [47]. They are effective against organisms with intrin- sic or acquired triazole resistance, can be safely used in Oral and oesophageal candidiasis patients with renal and hepatic insufficiency, and have few Oral and oesophageal candidiasis predominantly occurs in drug interactions. The minimum inhibitory concentrations patients with AIDS, but occasionally in other settings. of C. parapsilosis, C. guilliermondii and C. famata are While oral candidiasis can often be managed with topical higher than those of other species, but the clinical therapy (e.g. clotrimazole, miconazole, nystatin, ampho- relevance of this phenomenon is unclear [50,51]. Echino- tericin B lozenges), oesophageal candidiasis requires candin resistance resulting from amino acid substitutions systemic antifungal therapy. Fluconazole is safe and effec- within defined regions of the target protein Fks1 is increas- tive, and is the initial systemic treatment of choice. A ingly recognised, but is usually only observed following single dose of 400 mg is as effective as giving 150 mg for prolonged therapy [52,53] 14 days [62]. Challenges and opportunities for the treatment of dis- If there is resistance to fluconazole, the use of an seminated candidiasis are (i) improved diagnostics (earlier alternative triazole is possible, but a polyene or an echi- therapy is associated with improved mortality) [54]; (ii) nocandin is required if triazole cross-resistance is present potential advantage of immunisation [55]; and (iii) better [63–65]. Although echinocandins are effective agents for risk stratification, especially for failure of fluconazole pro- the treatment of oesophageal candidiasis, the proportion of phylaxis. patients that relapse tends to be higher compared with the triazoles. Echinocandin resistance can develop after pro- Candidiasis of the renal tract longed therapy, with mutations within Fks1 being respon- ThepresenceofCandida in the urine (candiduria) occurs sible. An orally bioavailable agent with activity against in  9% of hospitalised patients [56,57], and might be the triazole-resistant Candida spp. would be useful.

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Figure 2. Modes of action of the main classes of antifungal agents used to treat invasive fungal infections. (a) The active site of the triazoles contacts a haem moiety at the bottom of a binding pocket in the target fungal enzyme 14-a demethylase. The side chain (denoted by R) that is unique to each triazole is thought to be important for the positioning and orientation of the triazole within the binding pocket. (b) The polyenes bind to within the fungal cell membrane, leading to the formation of transmembrane pores that result in membrane depolarization and ultimately fungal cell death. (c) The echinocandins are nonreversible inhibitors of the enzyme glucan synthase that is responsible for the conversion of uridine 5 diphosphate to 1,3 b-D glucan. (d) Flucytosine (5-FC) is a subversive substrate within the pyrimidine salvage pathway. The normal pathway and the toxic fluorinated metabolites are shown. Fluorinated nucleotides lead to disruption of nucleic acid synthesis.

Vulvovaginal candidiasis Invasive aspergillosis Vulvovaginal candidiasis is common in pre-menopausal Invasive aspergillosis is associated with 50% mortality, women. Most patients respond promptly to antifungal despite a better understanding of pathogenesis and the therapy, but some have recurrent infection or persistent advent of new diagnostic and therapeutic methods. This symptoms. Women with cystic fibrosis are especially prone syndrome remains extremely difficult to treat, and is often to recurrent infection, probably because systemic antibac- diagnosed relatively late. The majority of patients are terial therapy disrupts the normal vaginal flora [66]. treated presumptively, without definitive microbiological Nystatincanbeeffectiveforthetreatmentofflucona- evidence. zole-resistant organisms (usually caused by C. glabrata), One of the significant therapeutic advances in the past but local application of a weak solution of boric acid or decade is an improved understanding of dose-response amphotericin B and flucytosine pessaries are alternatives relationships for the polyenes. Early studies suggested [67,68]. The triazoles are contraindicated in pregnancy that improved responses were possible using high dosages [69] and therefore, prolonged courses of triazoles can of amphotericin B deoxycholate (e.g. 1–1.5 mg kgÀ1) [72]. interfere with plans for conception in women of childbear- Despite few data, this approach was widely adopted. ing age with recurrent vulvovaginal candidiasis. During Unfortunately, however, these dosages are accompanied pregnancy, topical therapy is the only option. A systemic by significant toxicity, which is independently associated antifungal therapy for vulvovaginal candidiasis caused by with higher mortality, requirement for haemodialysis and C. glabrata andthatissafeinpregnancywouldbeparticu- excessive hospital costs [73,74]. larly useful. The lipid formulations of amphotericin B are less toxic than deoxycholate. Despite the ability to be able to admin- Treatment of infections caused by Aspergillus spp ister more active drug, there is no direct evidence that is a leading cause of life-threatening these preparations are more effective than amphotericin B mould infection in immunocompromised patients. Humans deoxycholate. Several clinical trials have enabled effective are continuously exposed to this environmentally ubiqui- dosages of liposomal amphotericin B to be identified tous mould. A. fumigatus is unique for the extent of clinical [11,12,75]. Although the optimal dosage of liposomal syndromes it produces, which range from allergic syn- amphotericin B is not known, most authors advocate dromes through chronic invasive forms and acute rapidly 3mgkgÀ1 for invasive aspergillosis. progressive life-threatening infection. Other species of Another major advance has been the introduction of Aspergillus cause some similar syndromes. Aspergillus triazoles with anti-Aspergillus activity. The efficacy and terreus and Aspergillus nidulans are resistant to ampho- safety of itraconazole for invasive aspergillosis was estab- tericin B. Triazole resistance has been documented in lished in an early noncomparative trial with approxi- environmental strains of A. fumigatus [70] and during mately 33% of patients responding to therapy [76]. treatment [71]. Voriconazole [followed by other licensed antifungal

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Review Trends in Microbiology Vol.xxx No.x therapy (OLAT)] is superior to amphotericin B deoxycho- and many patients have innate immunological defects [86– late (followed by OLAT) in terms of efficacy, improved 88]. An aspergilloma (fungal ball) within pulmonary cavities survival and the incidence of adverse events (53% versus can be intermittently present [84,85]. 32% response rate, respectively) [77]. Other studies sup- Most patients with chronic pulmonary aspergillosis port favourable outcomes of voriconazole therapy for require long-term maintenance antifungal therapy (i.e. patients with invasive aspergillosis [78,79]. Posaconazole months to years). Despite some limitations, the triazoles is effective for the treatment of patients who are refractory are the only oral agents available for this purpose [89].Of to or intolerant of other antifungal agents, but its use as a considerable concern is the recent emergence of triazole first line agent has not been established [38]. resistance [71,90]. Isolated resistance to itraconazole is The echinocandins have both in vitro and in vivo activity commonly observed, but cross-resistance to voriconazole against Aspergillus spp. [43]. Hyphae exposed to echinocan- and/or posaconazole occurs in >50% of instances. Infec- dins are gnarled, swollen and excessively branched, but tions by strains resistant to all triazoles require parenteral remain viable. The echinocandins produce a modest survi- therapy with amphotericin B or an echinocandin. val advantage in laboratory animal models of invasive The challenges to be overcome include: (i) the lack of pulmonary aspergillosis, despite the fungal burden in the knowledge of the optimal triazole regimen; and (ii) the fact being unchanged or paradoxically higher. In echino- that regimens developed for acute invasive disease might candin-treated laboratory animals, the fungi appear to have not achieve high concentrations within pulmonary cavities a reduced propensity for causing pulmonary injury [44]. and therefore might promote antifungal resistance. Oppor- Increasing evidence suggests that the echinocandins cause tunities for future therapy include: (i) the need to identify unmasking of immunoreactive fungal epitopes that might regimens that do not promote the generation of antifungal facilitate cooperative immunological killing [80–82]. Unfor- resistance; (ii) the development of orally bioavailable com- tunately, the optimal echinocandin dosage for invasive pounds without cross-resistance to the triazoles, which are aspergillosis is not known and is very difficult to determine suitable for long-term use; (iii) further definition of the role using current preclinical data. The echinocandins are poten- of adjunctive immunotherapy; (iv) rapid, direct methods to tially useful for salvage therapy, but their role in the identify triazole resistance, as cultures are often negative; primary treatment of invasive aspergillosis is less clear. and (v) strategies to minimise pulmonary fibrosis. There are several challenges in the treatment of invasive aspergillosis: (i) many diagnoses are established relatively Allergic bronchopulmonary aspergillosis and severe late, often when there has been significant tissue damage; asthma with fungal sensitisation (ii) the biomarkers with prognostic value that can be used to Patients with either allergic bronchopulmonary aspergillo- follow antifungal therapy in real time are only rudimentary; sis (ABPA) or severe asthma with fungal sensitisation (iii) there is often uncertainty about the diagnosis because of (SAFS) appear to benefit from itraconazole treatment the suboptimal specificity of current diagnostic methods; in [91–93]. However, approximately 15% of patients cannot many cases a syndrome that is consistent with an invasive tolerate itraconazole for months [93]. Relapses are common, fungal infection is assumed to be invasive aspergillosis and an interaction between itraconazole and inhaled corti- without any microbiological data available to enable a diag- costeroid often leads to higher reduced metabolism of corti- nosis to be established at either a genus or species level; (iv) costeroid, thus increasing corticosteroid side-effects. emerging triazole resistance. Therefore, the opportunities Because long-term therapy is required for both ABPA and are as follows: (i) development of improved diagnostics; (ii) SAFS, safe and well-tolerated drugs are essential. Although development of drugs that might have an effect upon patho- A. fumigatus is the most common fungus involved, others genesis (e.g. compounds that inhibit angioinvasion or pre- such as Alternaria alternata and Cladosporium herbarum vent dissemination); (iii) further study of the role of adjuvant are implicated, requiring a slightly different antifungal treatments (e.g. surgery, granulocyte transfusions and spectrum compared with agents required for invasive dis- immunotherapy); (iv) development of strategies that enable ease. Broad-spectrum, orally bioavailable, mould-active the attainment of near maximal antifungal effect as quickly agents would potentially enable more effective treatment as possible (e.g. innovative dosing strategies, therapeutic of patients with these allergic syndromes. drug monitoring and combination antifungal therapy); (iv) development of safe, orally bioavailable compounds with Cryptococcal meningitis broad spectrum anti-Aspergillus activity, which are devoid Cryptococcal meningitis occurs in a wide range of immu- of drug interactions and have a low propensity for devel- nocompromised patients with defects of cellular immunity. opment of resistance; and (v) development of prognostic The global burden of infection largely resides with patients biomarkers that enable real-time monitoring of antifungal with AIDS living in resource-poor settings. Patients with therapy. high infectious burden and raised intracranial pressure tend to have a worse prognosis. Induction therapy with Chronic pulmonary aspergillosis and aspergilloma amphotericin B deoxycholate 0.7–1.0 mg kgÀ1 combined Chronic pulmonary aspergillosis is a slowly progressive with flucytosine 100 mg kgÀ1 per day for 2 weeks, followed infection that is characterised by prominent constitutional by further consolidation therapy with fluconazole is con- and respiratory symptoms, progressive development of sidered standard care in the developed world [19]. Man- cavities in the lung, and the presence of anti-Aspergillus agement of raised intracranial pressure is important. IgG and IgE antibodies [83–85]. This syndrome invariably Recent studies suggest that the fungal burden in the occurs in the context of pre-existing structural lung disease, cerebrospinal fluid has prognostic significance, and can be

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Review Trends in Microbiology Vol.xxx No.x used as biomarker for antifungal therapy. This has facili- antifungal agents. Orally bioavailable and well-tolerated tated several clinical trials examining innovative regi- alternatives to the triazoles are urgently required. Several mens. The inability to administer amphotericin B possible antifungal product profiles are shown in Box 1 (not deoxycholate in some resource-poor settings has led to in priority order). Not all novel antifungal agents necess- attempts to optimise oral induction regimens. Fluconazole arily need to have a broad spectrum of activity, but excel- 1200 mg per day is more rapidly fungicidal than 800 mg, lent activity against either Candida or Aspergillus is but is still associated with 54% mortality at 10 weeks [94]. required for a significant economic return. Antifungal The addition of flucytosine to fluconazole 1200 mg per day therapy of fungal allergic syndromes, including eosinophi- results in greater fungicidal activity compared with fluco- lic fungal rhinosinusitis, requires further study. nazole monotherapy [95]. The effect of this combination in Several other requirements for further research are comparison with amphotericin B is not known, but further shown in Box 2. Accurate diagnostic tests with rapid turn studies are planned. around time will facilitate clinical trials. Triazole and The challenges and opportunities for the treatment of echinocandin resistance is of concern, and requires more cryptococcal meningitis are: (i) dramatically increased rapid approaches to detection and continued surveillance. levels of funding for this global health problem; (ii) further Understanding which patients are at highest risk of devel- refinement of optimal regimens for induction and main- oping a life-threatening infection is at present a major tenance therapy, including oral options and other innova- deficiency, and genetic markers will probably assist in risk tive regimens; (iii) development of new orally bioavailable assessment. Both combination antifungal therapy and drugs that penetrate the central nervous system; (iv) immunomodulatory approaches require detailed study to increasing the availability of oral flucytosine and other maximise survival, and minimise tissue damage and fibro- compounds in resource-poor settings; and (v) development sis. Despite major immunosuppression, vaccines to pre- of oral bioavailable formulations of amphotericin B. vent infection require study, and might be useful for improving therapeutic outcomes. Mucorales and other rare moulds The Mucorales cause life-threatening infection of the Conflict of interest statement sinuses, , brain, gut, skin and kidney. Only ampho- In the past 5 years, DD has had received grant support tericin B formulations and posaconazole are reliably from Astellas, Merck, Pfizer, F2G, OrthoBiotech, Indevus, active against these organisms. When combined with Basilea, the Fungal Research Trust, the Wellcome Trust, aggressive surgery, the response rates to these agents the Moulton Trust, The Medical Research Council, The is approximately 70% [39,96]. An understanding of Chronic Granulomatous Disease Research Trust, the optimal antifungal therapy for these infections has been National Institute of Allergy and Infectious Diseases hampered by difficulties in predicting the in vivo and the European Union. He has been an advisor/consult- responses to polyenes or posaconazole based on in vitro ant to Basilea, Vicuron (now Pfizer), Pfizer, Schering susceptibility tests and in developing suitable laboratory Plough (now Merck), Indevus, F2G, Nektar, Daiichi, animal models that are faithful mimics of human disease. Sigma Tau, Astellas, Gilead and York Pharma. He has The widespread use of voriconazole, which has no been paid for talks on behalf of Schering, Astellas, Merck, inherent activity, is associated with an increase in the Dainippon, AstraZeneca, Myconostica and Pfizer. He incidence of infection in some studies [97]. However, the holds founder shares in F2G Ltd and Myconostica Ltd, combination of a polyene and echinocandin might be both University of Manchester spin-out companies. WH effective [98]. Iron chelation therapy is another possible has been an advisor or consultant to Astellas, Pfizer, therapeutic option [99]. Gilead, Schering and Vectura. He has received research There are increasing reports of other moulds that cause support from Basilea, Astellas, Gilead and, Schering, and devastating infections in a range of patients, including has been paid for talks on behalf of Pfizer, Gilead, Astellas dematiaceous (e.g. Curvularia and Bipolaris) [100] and and Merck. hyaline moulds (e.g. Fusarium). These infections are likely to remain problematic unless new active agents active Acknowledgements against common yeasts and moulds incidentally have an WH is funded by a National Institute of Health Research (NIHR) extended spectrum of activity. Clinician Scientist Fellowship. We are indebted to Elaine Bradley of UHSM who drew the figures. Challenges and opportunities include: (i) development of international networks that enable collective experience References of these infections and facilitation of clinical trials; (ii) the 1 Park, B.J. et al. (2009) Estimation of the current global burden of development of laboratory animal models that enable in cryptococcal meningitis among persons living with HIV/AIDS. Aids vivo-to-human bridging studies that can be used to identify 23, 525–530 suitable regimens in the absence of large randomised 2 Agarwal, R. et al. (2009) Aspergillus hypersensitivity and allergic bronchopulmonary aspergillosis in patients with bronchial asthma: clinical trial data; (iii) development of adjunctive treat- systematic review and meta-analysis. Int. J. Tuberc. Lung Dis. 13, ments (e.g. iron chelation); and (iv) further development of 936–944 novel antifungal combinations. 3 Vicente, M.F. et al. 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